Research will continue on the long term goal of elucidating the structural and mechanistic bases of ?-crystallin chaperone activity, defining its contribution to the maintenance of lens transparency and understanding its role in the development of cataract. Cataract is the leading cause of blindness world-wide and its treatment remains a significant burden to the US health care system. Aging degrades lens optical properties through modifications of lens proteins which compromise their stability, promote their aggregation and disturb the protein order required for refractivity and transparency. The dominant model in the field hypothesizes a central role for the lens small heat-shock protein ?-crystallin in delaying the onset of aggregation through recognition and binding of destabilized lens proteins. Despite 20 years of progress in identification of age-related modifications and an expansive effort to define ?-crystallin chaperone mechanism, there remains a divide between in vitro models of lens protein interactions and congenital and age-related cataract phenotypes. The proposed research plan will attempt, for the first time, to challenge mechanistic in vitro hypotheses and models of lens protein stability and ?-crystallin chaperone function in the intact lens of zebrafish (Danio rerio). An outstanding team of collaborators has been recruited to enable the successful completion of this multidisciplinary plan. Aim 1 will continue our productive studies of ?-crystallin structure and mechanism. We will test if a structural model of ?-crystallin activation, developed in the previous funding period, explains the evolutionary variation in chaperone properties and the loss of binding regulation in mutants of ?-crystallin associated with human autosomal dominant hereditary cataracts. We will use spectroscopy to investigate the structural and energetic aspects of the chaperone interaction between ?- and ?-crystallin. Aim 2 proposes to generate transgenic zebrafish models to determine how ?-crystallin chaperone affinity is shaped by the crowded environment of the lens fiber cells and whether cataract phenotypes can be rescued by engineered ?-crystallin with designed functional properties.